The "free radical theory of oxygen toxicity" links the deleterious pulmonary effects of hyperoxia, cellular oxygen metabolism and the respiratory burst of activated inflammatory cells to highly reactive metabolic products of oxygen. These reactive oxygen species (ROS) can severely alter mitochondrial membrane potential and function, inactivate cellular enzymes, damage DNA, and destroy lipid bilayers leading to either cellular necrosis or apoptosis. To protect cells from these cytotoxic oxygen metabolites, a system of cooperative antioxidants have evolved with the primary defense being the superoxide dismutases (SODs). It has been repeatedly shown that induction or over-expression of the mitochondrial manganese SOD (MnSOD) can provide an effective, cytoprotective antioxidant defense which is strongly associated with lung cell's tolerance to superoxide induced injury and survival. Therefore, elucidating the lung's normal molecular mechanisms for stimulating endogenous antioxidant defenses by induction of MnSOD will lead to logical steps in the development of therapeutic regimens that are effective in preventing or ameliorating free radical mediated pulmonary toxicity. To this end, the goals of this proposal are to delineate the molecular mechanisms which control stimulus-dependent MnSOD gene expression through the action of a novel, cytokine-dependent, intronic enhancer element. AIMs I and III of this proposal focus on the identification of trans-acting regulatory factors and their co-activator protein partners using yeast One- and Two-Hybrid library screening, respectively. AIM II proposes a three pronged approach to verify in cells that potential enhancer-specific regulatory factors are in fact responsible for stimulus-dependent enhancer function. These include: functional evaluation by over-expression and dominant negative analysis; chromatin immunoprecipitation (ChIP); and PIN*POINT (ProteIN POsition Identification with Nuclease Tail) analysis. AIM IV will evaluate the MnSOD enhancer as a potential lung gene therapy tool in an in vivo model of lipopolysaccharide (LPS)-induced endotoxemia due to this element's inherent ability to respond to endogenous pro-inflammatory signals.